Extracts from the leaves of the Maiden hair tree or Ginkgo biloba have long been known to possess medicinal properties. Recently, Ginkgo biloba extracts (GBE) have been reported for use in treating asthma, tinnitus, impotence, immunosupression and memory loss among others. However, the clinical effect of GBE that is best documented is that of a vasodilator. For this reason, GBE is used as an aid to circulation and as an enhancer of memory and cognitive function in mammals. Over the counter tablets, which are taken orally, are the most commonly available form of GBE.
The primary constituents of GBE are compounds known as flavone glycosides. Secondary components in GBE include other flavonoids, ginkgo terpene lactones (ginkgolides and bilobalide), proanthocyanidines and undesired lipophilic constituents which contain alkyl bilobalide phenols. These components are typically present in the commercial GBE products currently available. GBE products must contain at least 24% by weight of ginkgo flavone glycosides, 6% by weight of ginkgo terpene lactones, and a maximum level of ginkgolic acid of 5 ppm in order to meet the standards adopted by the German Federal Institute for Drugs and Medical Devices and by U.S. manufacturers of phytomedicines.
The alkyl phenols constituents of GBE have been reported to be responsible for phenols, GBE, allergic reactions and skin irritations suffered by individuals that ingest GBE. More specifically, 2-hydroxy-6-alkyl benzoic acids where alkyl is equal to normal C.sub.13 to normal C.sub.19 ; 2-hydroxy-6-alkenyl-benzoic acids where alkenyl is equal to normal C.sub.13 to normal C.sub.19 with the requisite alkenyl double bond(s) in varying positions along the alkenyl chain; and 2-hydroxy-6-alkenyl-benzoic acids where alkenyl is equal to normal C.sub.13 to normal C.sub.19 with the requisite triple bond(s) in various positions along the alkynyl chain; are thought to be responsible for such reactions and irritations. Representative examples of these compounds are 6-pentadecyl-2-hydroxybenzoic acid, 6-(8-pentadecenyl)-2-hydroxybenzoic acid (known as ginkgolic acid) and 6-(8-pentadecynyl)-2-hydroxybenzoic acid. In particular, high levels of these compounds (exceeding 100 ppm of the GBE) are believed to be responsible for inducing allergic contact reactions of mucous membranes such as cheilitis, stomatitis, proctitis, and pruritus ani or skin irritations such as contact dermatitis. Thus, producers of GBE have sought a method for making an economical, clinically potent GBE that has low levels of the aforementioned allergenic alkyl phenols and which therefore avoids these allergic reactions.
The main alkyl-phenol component of GBE is ginkgolic acid (2-hydroxy-6-(trans-8-pentadecenyl)benzoic acid) which possesses a normal C.sub.15 side chain having a trans double bond at the C.sub.8 position. As employed herein the term ginkgolic acid derivatives refer to any 2-hydroxy-6-alkyl, alkenyl or alkynyl benzoic acid having a carbon chain length varying from normal C.sub.13 to normal C.sub.19. Included in this definition of ginkgolic acid derivatives are the ginkgols which are 3-alkyl phenol derivatives.
The method of the present invention is used to produce GBE in which the amount of these compounds is reduced to less than 5 ppm. Several methods exist for removing the allergenic alkyl phenols from GBE (See, for example, U.S. Pat. No. 5,637,302 and European Patent 0 477-968). The methods for removing alkyl phenols disclosed in these patents utilize expensive, environmentally unfriendly organic solvents such as n-butanol, toluene, n-hexane, and n-heptane as extraction agents. Apart from their high cost (which is a significant drawback), these techniques are impractical for economical large scale production of GBE due to the large volume and toxicity of the solvents needed to perform the extractions.
The present invention overcomes these problems by providing an efficient and environmentally friendly process amenable to economical large scale production of pharmaceutical grade GBE that is essentially free of ginkgolic acid and ginkgolic acid derivatives.
The present invention employs a supercritical fluid extraction step. As used herein, the term supercritical fluid means a gas existing above its critical temperature and pressure as defined in its phase diagram. When a gas such as CO.sub.2 is compressed, above its critical temperature, its density greatly increases, so the gas has the density of a liquid and the diffusivity of a gas. This makes suprecritical fluids superior solvents. The solvent properties of supercritical CO.sub.2 can be attenuated by changing the pressure at which the extraction is performed. Typical ranges of temperature and pressure for supercritical CO.sub.2 extractions which are useful in the present invention are between about -10 and about 180.degree. C. and between about 100 and about 350 atm (see, for example, U.S. Pat. No. 4,466,923).